Neuronal enhancers get a break
2021; Cell Press; Volume: 109; Issue: 11 Linguagem: Inglês
10.1016/j.neuron.2021.05.008
ISSN1097-4199
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoEmerging evidence shows that neuronal DNA is continuously broken and repaired in a non-random fashion within the genome. Two recent studies, Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar and Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar, use sequencing of newly synthesized DNA in post-mitotic neurons to map hotspots of DNA repair across the genome. Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar further show that the repair sites are associated with single-stranded DNA breaks that predominantly occur on neuronal enhancers at sites of CpG methylation. Emerging evidence shows that neuronal DNA is continuously broken and repaired in a non-random fashion within the genome. Two recent studies, Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar and Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar, use sequencing of newly synthesized DNA in post-mitotic neurons to map hotspots of DNA repair across the genome. Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar further show that the repair sites are associated with single-stranded DNA breaks that predominantly occur on neuronal enhancers at sites of CpG methylation. Post-mitotic neurons can last the entire lifespan of an organism and must rely on efficient DNA repair mechanisms to maintain the integrity of their genomes. Consequently, mutations in DNA repair pathway components lead to neurodevelopment and neurodegenerative diseases (Chow and Herrup, 2015Chow H.M. Herrup K. Genomic integrity and the ageing brain.Nat. Rev. Neurosci. 2015; 16: 672-684https://doi.org/10.1038/nrn4020Crossref PubMed Scopus (110) Google Scholar). Surprisingly, in addition to DNA damage from environmental insults and transcription-associated DNA breaks, neurons actively break their DNA during neuronal activity, further increasing the DNA repair burden of neurons (Suberbielle et al., 2013Suberbielle E. Sanchez P.E. Kravitz A.V. Wang X. Ho K. Eilertson K. Devidze N. Kreitzer A.C. Mucke L. Physiologic brain activity causes DNA double-strand breaks in neurons, with exacerbation by amyloid-β.Nat. Neurosci. 2013; 16: 613-621https://doi.org/10.1038/nn.3356Crossref PubMed Scopus (293) Google Scholar; Madabhushi et al., 2015Madabhushi R. Gao F. Pfenning A.R. Pan L. Yamakawa S. Seo J. Rueda R. Phan T.X. Yamakawa H. Pao P.C. et al.Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes.Cell. 2015; 161: 1592-1605https://doi.org/10.1016/j.cell.2015.05.032Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Thus, it is imperative to understand how neurons maintain genome integrity by efficiently distributing DNA repair resources. It is well established that DNA repair is associated with varying degree of DNA synthesis depending upon the nature of the damage and choice of repair pathway (Chatterjee and Walker, 2017Chatterjee N. Walker G.C. Mechanisms of DNA damage, repair, and mutagenesis.Environ. Mol. Mutagen. 2017; 58: 235-263https://doi.org/10.1002/em.22087Crossref PubMed Scopus (567) Google Scholar). Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar used sequencing of EdU-incorporated DNA to identify locations of DNA synthesis associated with DNA repair, which they termed synthesis-associated with repair sequencing (SAR-seq). Given the lack of genome replication in post-mitotic neurons, any EdU incorporation can be attributed to DNA repair and is similar to the approach used in a concurrent study by Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar (termed Repair-seq) and also a previous approach used to map DNA repair synthesis during mitosis (Macheret et al., 2020Macheret M. Bhowmick R. Sobkowiak K. Padayachy L. Mailler J. Hickson I.D. Halazonetis T.D. High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing.Cell Res. 2020; 30: 997-1008https://doi.org/10.1038/s41422-020-0358-xCrossref PubMed Scopus (33) Google Scholar). To map the locations of DNA repair, Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar labeled post-mitotic induced pluripotent stem cell (iPSC)-derived glutamatergic neurons with the thymide analog EdU for 18 h, followed by biotinylation of the incorporated EdU, fragmentation and affinity purification of the biotinylated DNA fragments, and high-throughput sequencing (Figure 1). SAR-seq revealed more than 55,000 highly reproducible peaks across the genome. Interestingly, while SAR-seq in rat cortical neurons revealed similar results, iPSC-derived skeletal muscle cells and G0-arrested pre-B cells failed to produce a signal, demonstrating that recurrent locations of DNA repair could be unique to neurons. Comparison of SAR-seq signal to regions of the chromatin with increased accessibility as measured by assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that DNA-repair-associated synthesis is enriched in open chromatin. Interestingly, comparing the SAR-seq profile with neuronal enhancers identified by chromatin immunoprecipitation (ChIP-seq) of H3K4me1, H3K27Ac, and MLL4 revealed that the enrichment of DNA repair at active chromatin was dominated by enhancers compared to other regions of the active chromatin such as promoters and non-enhancer elements. Further, SAR-seq signal was not enriched at iPSC-specific enhancer locations, indicating that the overlap between SAR-seq and enhancers was restricted to enhancers active in the neurons. Indeed, it was found that 90% of super-enhancers that define neuronal identity had SAR-seq peaks in contrast to only 25% for conventional neuronal enhancers. Together, these results demonstrate that neuronal enhancers are hotspots for recurrent DNA repair activity. It is interesting to note that the study by Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar found enrichment of DNA repair hotspots at short promoters (<1 kb), 5′ untranslated regions, and gene bodies rather than enhancers. However, both studies converged on the exact same motif enrichment at repair sites corresponding to the ONECUT family of transcription factors. The discrepancy in genomic enrichment annotation of repair sites between the two studies might be due to the lack of a direct comparison between repair sites and enhancer marks, such as H3K4me1, in the study by Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar. Because DNA synthesis is a ubiquitous aspect of many DNA repair pathways, the next obvious question was the nature of DNA damage occurring at the neuronal enhancers. END-seq, a method for directly detecting double-strand breaks (DSBs), revealed that unchallenged neurons did not have DSBs at the locations of SAR-seq peaks. Single- and double-stranded DNA breaks (SSBs and DSBs, respectively) activate poly(ADP-ribose) polymerases (PARPs) that modify local proteins with poly(ADP-ribose) indicating the presence of these lesions. In addition, PARPs recruit XRCC1 protein to SSBs to mediate its repair (Chatterjee and Walker, 2017Chatterjee N. Walker G.C. Mechanisms of DNA damage, repair, and mutagenesis.Environ. Mol. Mutagen. 2017; 58: 235-263https://doi.org/10.1002/em.22087Crossref PubMed Scopus (567) Google Scholar). Comparing ADP-ribosylation locations and XRCC1 binding sites using ChIP-seq to SAR-seq signals revealed a striking overlap, indicating that the DNA repair at enhancers were due to SSBs. To further validate this, Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar devised a clever method to detect SSBs directly by first converting SSBs to DSBs by treating the neurons with the single-strand-specific S1 nuclease and then detecting the DSBs using END-seq (Figure 1). However, this method failed to detect the endogenous SSBs expected at the neuronal enhancers. The authors hypothesized that the failure of S1 nuclease END-seq was due to the rapid repair of SSBs before the S1 nuclease had a chance to cleave the second strand. To circumvent this, SSB repair was inhibited by the addition of chain-terminating dideoxynucleotides (ddNTPs) before performing S1 nuclease END-seq. This modification allowed the precise detection of endogenous SSBs and revealed extensive colocalization between SSBs and locations of DNA synthesis by SAR-seq. Further, the base-pair resolution afforded by S1 nuclease END-seq revealed that, within the enhancers, SSBs were enriched at C/G nucleotides. SSBs can be resolved by both short-patch and long-patch repair pathways. In a short-patch repair pathway, a single nucleotide at the break site is replaced by DNA polymerase β (Polβ) recruited by XRCC1. In a long-patch repair, 2–20 nucleotides are replaced by POLε and POLδ (Chatterjee and Walker, 2017Chatterjee N. Walker G.C. Mechanisms of DNA damage, repair, and mutagenesis.Environ. Mol. Mutagen. 2017; 58: 235-263https://doi.org/10.1002/em.22087Crossref PubMed Scopus (567) Google Scholar). To investigate the contribution of these distinct pathways to SAR-seq signal, Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar performed SAR-seq after pharmacological and CRISPRi-based inhibition of either short-patch or long-patch repair. These experiments demonstrated that the inhibition of short-patch repair consistently increased the SAR-seq signal, whereas inhibition of the long-patch repair decreased the SAR-seq signal. Because SAR-seq signal is directly proportional to the amount of EdU incorporation, these results show that both short- and long-patch repair resolve SSBs in neurons; however, SAR-seq signal primarily originates from the long-patch repair pathway. Interestingly, this raises the question of whether SAR-seq is sensitive at detecting DSBs. Post-mitotic cells like neurons primarily resolve DSBs by non-homologous end joining, and highly localized DSBs induced by neuronal activity might involve DNA synthesis below the threshold of detection. Indeed, Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar showed that Repair-seq could not detect activity-dependent DSBs after neuronal stimulation (Suberbielle et al., 2013Suberbielle E. Sanchez P.E. Kravitz A.V. Wang X. Ho K. Eilertson K. Devidze N. Kreitzer A.C. Mucke L. Physiologic brain activity causes DNA double-strand breaks in neurons, with exacerbation by amyloid-β.Nat. Neurosci. 2013; 16: 613-621https://doi.org/10.1038/nn.3356Crossref PubMed Scopus (293) Google Scholar; Madabhushi et al., 2015Madabhushi R. Gao F. Pfenning A.R. Pan L. Yamakawa S. Seo J. Rueda R. Phan T.X. Yamakawa H. Pao P.C. et al.Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes.Cell. 2015; 161: 1592-1605https://doi.org/10.1016/j.cell.2015.05.032Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar demonstrated that treatment of neurons with Etoposide that generate widespread DSBs at locations of active transcription resulted in SAR-seq signal within gene bodies. Finally, Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar explored the potential sources of these SSBs. Given the enrichment of SSBs at C/G nucleotides and the high prevalence of CpG methylation in post-mitotic neurons compared with other cell types, the authors suspected that SSBs could be a consequence of cytosine demethylation. Consistent with this hypothesis, there was a striking correlation between genome-wide SSB profiles and distribution of cytosine demethylation intermediates 5fc and 5 hmc in neurons. Indeed, Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar also found a close association between DNA repair hotspots and sites of CpG methylation. The study by Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar raises several important questions regarding the source and consequences of SSBs at neuronal enhancers. One intriguing question is whether recurrent SSBs at enhancers have any functional relevance. Previous studies have shown that DSBs can regulate transcription in both neurons and peripheral cells (Ju et al., 2006Ju B.G. Lunyak V.V. Perissi V. Garcia-Bassets I. Rose D.W. Glass C.K. Rosenfeld M.G. A topoisomerase IIbeta-mediated dsDNA break required for regulated transcription.Science. 2006; 312: 1798-1802https://doi.org/10.1126/science.1127196Crossref PubMed Scopus (638) Google Scholar; Madabhushi et al., 2015Madabhushi R. Gao F. Pfenning A.R. Pan L. Yamakawa S. Seo J. Rueda R. Phan T.X. Yamakawa H. Pao P.C. et al.Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes.Cell. 2015; 161: 1592-1605https://doi.org/10.1016/j.cell.2015.05.032Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). While Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar did not find any association between SAR-seq signal intensity and transcript levels, Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar found a correlation between total Repair-seq reads and transcription, but not with recurrent peaks of repair. Activity-regulated neuronal enhancers have been shown to be sites of wide-spread transcription producing so-called enhancer RNAs (Kim et al., 2010Kim T.K. Hemberg M. Gray J.M. Costa A.M. Bear D.M. Wu J. Harmin D.A. Laptewicz M. Barbara-Haley K. Kuersten S. et al.Widespread transcription at neuronal activity-regulated enhancers.Nature. 2010; 465: 182-187https://doi.org/10.1038/nature09033Crossref PubMed Scopus (1628) Google Scholar). It will be interesting to test whether SSBs at enhancers have the potential to regulate enhancer RNA transcription. However, the most obvious question raised by the study by Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar is the relevance of recurrent SSBs on genome stability and maintenance. One prediction from this finding is that repeated break and repair cycles over the lifetime of neurons would lead to the accumulation of mosaic somatic nucleotide variation (SNVs) in an age-dependent manner during normal aging. Consistent with this prediction, single-cell measurements of mosaic SNVs in neurons from three human brains revealed an enrichment of methylated cytosine to thymidine mutations (Lodato et al., 2015Lodato M.A. Woodworth M.B. Lee S. Evrony G.D. Mehta B.K. Karger A. Lee S. Chittenden T.W. D'Gama A.M. Cai X. et al.Somatic mutation in single human neurons tracks developmental and transcriptional history.Science. 2015; 350: 94-98https://doi.org/10.1126/science.aab1785Crossref PubMed Scopus (329) Google Scholar). Another interesting hypothesis is that DNA repair hotspots could indicate locations in the genome that are disproportionately more protected. Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar show that constrained elements in the human genome identified by genomic evolutionary rate profiling are enriched for DNA repair hotspots. Consistent with this, the most stable repair hotspots upon treatment with DNA damaging agents were also at constrained elements. Future studies from a larger sample size and wider representation of age groups will be required to establish the significance of recurrent DNA repair at enhancers and other DNA repair hotspots. Finally, mutations in the SSB repair pathway components could tip the scale of mutational burden at neuronal enhancers, causing a shift from normal age-related decline in function to age-related diseases of the brain. In conclusion, the studies by Wu et al., 2021Wu W. Hill S.E. Nathan W.J. Paiano J. Callen E. Wang D. Shinoda K. van Wietmarschen N. Colón-Mercado J.M. Zong D. et al.Neuronal enhancers are hotspots for DNA single-strand break repair.Nature. 2021; https://doi.org/10.1038/s41586-021-03468-5Crossref Scopus (36) Google Scholar and Reid et al., 2021Reid D.A. Reed P.J. Schlachetzki J.C.M. Nitulescu I.I. Chou G. Tsui E.C. Jones J.R. Chandran S. Lu A.T. McClain C.A. et al.Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.Science. 2021; 372: 91-94https://doi.org/10.1126/science.abb9032Crossref PubMed Scopus (18) Google Scholar reveal a new paradigm in neuronal genome integrity with significance to both normal aging and age-related diseases.
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